The goal of this competitive renewal is to determine how bile acids are transported intracellularly in the liver. We will test the hypothesis that: The intracellular bile carrier in hepatocytes is a microsomal membrane protein, mP52, that shares epitopes with the 48kD Na+-dependent bile acid carrier in basolateral plasma membranes, for which it may also be a precursor. We will therefore determine the protein structure, functions and biogenesis of MP52, for comparison with the 48kD Na+-dependent transporter and other organic anion transporters of liver plasma membranes. 1) mP52 will be isolated from microsomal membranes by detergent solubilization and bile acid affinity chromatography. Amino acid sequence analysis will be done, and if we confirm our preliminary data that shows difference from the sequence of the 48kD protein, we will screen rat liver CDNA library in order to determine the entire sequence of mP52 and do studies of mP52 gene expression and its constituted in proteoliposomes, to verify its transport function. 2) We will study the biogenesis of mP52 and the 48kD protein by pulse- chase labelling and subcellular fractionation in vivo and in isolated hepatoytes. We will use the biogenesis of the polymeric IgA receptor and the canalicular glycoprotein GP110, as models for these studies. We will also use a validated subcellular fractionation protocol to isolate vesicles of the transcytotic pathway to look for intracellular vesicles that carry mP52. Knowing the structural basis of the intracellular transport processes will extend our understanding of how the liver transports bile acids and organic anions in health, and how these processes are deranged in liver disease.